Luminogenic and nonluminogenic multiplex assay

ABSTRACT

A method to detect the presence or amount of at least one molecule for an enzyme-mediated reaction in a multiplex luminogenic/nonluminogenic assay is provided.

BACKGROUND OF THE INVENTION

Luminescence is produced in certain organisms as a result of aluciferase-mediated oxidation reaction. Luciferase genes from a widevariety of vastly different species, particularly the luciferase genesof Photinus pyralis and Photuris pennsylvanica (fireflies of NorthAmerica), Pyrophorus plagiophthalamus (the Jamaican click beetle),Renilla reniformis (the sea pansy), and several bacteria (e.g.,Xenorhabdus luminescens and Vibrio spp), are extremely popularluminescence reporter genes. Firefly luciferase is also a popularreporter for determining ATP concentrations, and, in that role, iswidely used to detect biomass. Luminescence is also produced by otherenzymes when those enzymes are mixed with certain synthetic substrates,for instance, alkaline phosphatase and adamantyl dioxetane phosphate, orhorseradish peroxidase and luminol.

Luciferase genes are widely used as genetic reporters due to thenon-radioactive nature, sensitivity, and extreme linear range ofluminescence assays. For instance, as few as 10⁻²⁰ moles of fireflyluciferase can be detected. Consequently, luciferase assays of geneactivity are used in virtually every experimental biological system,including both prokaryotic and eukaryotic cell cultures, transgenicplants and animals, and cell-free expression systems. Similarly,luciferase assays used to determine ATP concentration are highlysensitive, enabling detection to below 10⁻¹⁶ moles.

Luciferases can generate light via the oxidation of enzyme-specificsubstrates, e.g., luciferins. For firefly luciferase and all otherbeetle luciferases, light generation occurs in the presence of magnesiumions, oxygen, and ATP. For anthozoan luciferases, including Renillaluciferase, only oxygen is required along with the substratecoelentrazine. Generally, in luminescence assays to determine geneticactivity, reaction substrates and other luminescence activating reagentsare introduced into a biological system suspected of expressing areporter enzyme. Resultant luminescence, if any, is then measured usinga luminometer or any suitable radiant energy-measuring device. The assayis very rapid and sensitive, and provides gene expression data quicklyand easily, without the need for radioactive reagents.

Luciferases are one of a number of reporters, e.g., firefly luciferase,Renilla luciferase, chloramphenicol acetyl transferase (CAT),beta-galactosidase (lacZ), beta-glucuronidase (GUS) and variousphosphatases, such as secreted alkaline phosphatase (SEAP) anduteroferrin (Uf; an acid phosphatase), that have been combined and usedas co-reporters of genetic activity. A dual enzyme reporter systemrelates to the use, expression, and measurement of two individualreporter enzymes within a single system. In genetic reporting, dualreporter assays are particularly useful for assays in individual cellsor cell populations (such as cells dispersed in culture, segregatedtissues, or whole animals) genetically manipulated to simultaneouslyexpress two different reporter genes. Most frequently, the activity ofone gene reports the impact of the specific experimental conditions,while the activity of the second reporter gene provides an internalcontrol by which all sets of experimental values can be normalized. Dualenzyme reporter technology can also be employed with cell-freereconstituted systems such as cellular lysates derived for thesimultaneous translation, or coupled transcription and translation, ofindependent genetic materials encoding experimental and control reporterenzymes. Immunoassays may, likewise, be designed for dual reporting ofboth experimental and control values from within a single sample.

The performance of any dual enzyme reporter assay is based on thecharacteristics of the constituent enzyme chemistries and the ability tocorrelate their respective resulting data sets. Disparate enzymekinetics, assay chemistries and incubation requirements of variousreporter enzymes can complicate combining two reporter enzymes into anintegrated, single tube or well dual reporter assay format. One approachto integration of a dual reporter assay is described in U.S. Pat. No.5,744,320, which discloses particular general or specific quenchingagents for beetle and Renilla luciferase assays and demonstrates anexemplary dual reporter assay for sequentially determining luminescencefrom firefly luciferase then Renilla luciferase. Similarly, U.S. Pat.No. 6,586,196 discloses several dual reporter assay systems. Like thedual reporter systems disclosed in the '320 patent, luminescence is themeasurable product of each of two reactions in the '196 patent.Approaches to multiplexing of reporter assays which incorporate not onlydifferent substrates but also different detection technologies aredescribed in Liu et al. (2000) and Qazi et al. (2002). For instance, Liuet al. report luciferase and GFP activity in the same organism, whereenzyme activity is determined via luminescence and fluorescencedetection, respectively, in a stepwise fashion.

Reporters are also useful to detect the presence or activity ofmolecules within cells or supernatants. For instance, proteasesconstitute a large and important group of enzymes involved in diversephysiological processes such as protein turnover in blood coagulation,inflammation, reproduction, fibrinolysis, and the immune response.Numerous disease states are caused by, and can be characterized by, thealterations in the activity of specific proteases and their inhibitors.The ability to measure these proteases in research or in a clinicalsetting is significant to the investigation, treatment and management ofdisease states. For example, caspase-3 and caspase-7 are members of thecysteine aspartyl-specific protease (also known as the aspartatespecific-cysteine protease, “ASCP”) family and play key effector rolesin cell death in mammalian cells (Thornberry et al., 1992; Nicholson etal., 1995; Tewari et al., 1995; and Fernandes-Alnemri et al., 1996).

Proteases, however, are not easy to assay with their naturally occurringsubstrates. Moreover, many currently available synthetic substrates areexpensive, insensitive, and nonselective.

Numerous chromogenic and fluorogenic substrates have been used tomeasure proteases (Monsees et al., 1994; Monsees et al., 1995) andmodified luciferins have provided alternatives to fluorescent indicators(U.S. Pat. Nos. 5,035,999 and 5,098,828). Methods for using modifiedluciferins with a recognition site for a hydrolase as a pro-substratewere first described by Miska and Geiger (1989), where heterogeneousassays were conducted by incubating a modified luciferin with ahydrolase for a specified period of time, then transferring an aliquotof the mixture to a solution containing luciferase. Masuda-Nishimura etal. (2000) reported the use of a single tube (homogeneous) assay whichemployed a β-galactosidase substrate-modified luciferin.

Fluorescent or luminescent substrates or products of enzyme reactionshave been employed in protein assay multiplexing. For example,fluorescent beads having ligands for up to 15 different cytokines wereemployed to detect two or more different cytokines (DeJager et al.,2003) and fluorescein diphosphate and casein BODIPY-FL were employed todetect alkaline phosphatase and certain proteases (Nolkrantz et al.,2002).

However, what is needed is an improved assay, e.g., a homogeneous assay,to detect two or more proteins using different detection techniques.

SUMMARY OF THE INVENTION

The invention provides multiplexing of nonluminogenic, e.g., fluorescentor colorimetric, and luminogenic assays, e.g., in the same well, todetect the amount (e.g., activity) or presence in a sample of one ormore moieties, including cofactors for enzymatic reactions such as ATP,proteins (peptides or polypeptides) that bind to and/or alter theconformation of a molecule, e.g., proteins that modify or cleave apeptide or polypeptide substrate, or a molecule which is bound by and/oraltered by a protein. As used herein, a “luminogenic assay” includes areaction in which a first molecule, e.g., a peptide or polypeptidesubstrate for a first enzyme, the product of a reaction between thefirst molecule and an appropriate (first) protein, and/or a product of areaction between a different protein and the product of the firstreaction, is luminogenic. Thus, a luminogenic assay may directly orindirectly detect, e.g., measure, the amount or presence of a cofactorfor a reaction, a molecule which is bound by and/or altered by aprotein, or the protein. For instance, in one embodiment, a beetleluciferase and an appropriate luciferin substrate may be employed in aluminogenic assay to detect ATP concentration, while in anotherembodiment a substrate for a luciferase, which is modified to contain aprotease recognition site (modified, for example, via a covalent bond),may be employed in a luminogenic assay to detect the protease, i.e.,when luciferase is present. Luminogenic assays include chemiluminescentand bioluminescent assays including but not limited to those whichemploy or detect luciferase, β-galactosidase, β-glucuronidase,β-lactamase, a protease, alkaline phosphatase, or peroxidase, andsuitable corresponding substrates, e.g., modified forms of luciferin,coelenterazine, luminol, peptides or polypeptides, dioxetanes,dioxetanones, and related acridinium esters. As used herein, a“luminogenic assay reagent” includes a substrate, as well as acofactor(s) or other molecule(s) such as a protein, e.g., an enzyme, fora luminogenic reaction. In one embodiment, the luminogenic assay reagentmay be Z-DEVD-aminoluciferin, Z-LETD-aminoluciferin,Z-LEHD-aminoluciferin, or may be other substrates, e.g., peptide orpolypeptide substrates, linked to aminoluciferin, dihydroluciferin,luciferin 6′ methylether, or luciferin 6′ chloroethylether. Aluminogenic assay is one in which a luminogenic reaction yields at least1%, e.g., at least 10%, more light than a corresponding nonluminogenicassay.

A “nonluminogenic assay” includes a reaction in which a first molecule,e.g., a protein (a peptide or polypeptide), a (first) product of areaction between the first molecule and a suitable (first) protein(peptide or polypeptide), or a product of a reaction between a differentprotein and the first product is/are not luminogenic but may beotherwise detectable, e.g., the substrate and/or product(s) are detectedusing a fluorescent or colorimetric assay, which directly or indirectlymeasures the amount or presence of a cofactor for the reaction, themolecule or the protein which interacts with the molecule. For instance,a substrate for an enzyme may be modified to contain a fluorophore thatemits light of a certain wavelength only after the enzyme reacts withthe substrate and the fluorophore is contacted with light of a certainwavelength or range of wavelengths, e.g., (Z-DEVD)₂-rhodamine-110 is asubstrate for a caspase, and cleavage of that substrate by the caspasemay be monitored via fluorescence of rhodamine-110. As used herein, a“fluorogenic assay reagent” includes a substrate, as well as acofactor(s) or other molecule(s), e.g., a protein, for a fluorogenicreaction. A nonluminogenic assay is one in which a nonluminogenicreaction yields less than about 10%, e.g., less than about 1% or less,the luminescent signal of a corresponding luminogenic assay.

In one embodiment, molecules employed in the assays of the invention,e.g., those which bind and/or are altered by a protein, include onesthat are modified to contain a reporter molecule, i.e., a molecule whichis detectable or capable of detection, e.g., after one or moresubsequent reactions. For example, in one embodiment, a substrateemployed in a luminogenic assay of the invention includes a substratefor an enzyme to be detected, which substrate is covalently linked to asubstrate for a luminogenic reaction, while in another embodiment asubstrate employed in a fluorogenic assay may include a substrate for anenzyme to be detected, which substrate is covalently linked to one ormore fluorophores. In some embodiments, the molecule which is bound byand/or altered by a protein does not contain a reporter molecule.

As described herein, the amount or presence of more than one protease ina sample was detected using at least two different substrates, one whichhad a luminescent readout and one or more of which had a fluorescentreadout. For example, detection of a low abundance cellular protease wasachieved using a more sensitive luminescent approach, e.g., detection ofcaspase-8 with the substrate Z-LETD-aminoluciferin, followed by adetection of another protease using another substrate, for instance,caspase-3 with (Z-DEVD)₂-rhodamine-110. This assay thus combines thestrengths of both a fluorogenic reagent and the sensitivity of aluciferase-mediated luminescent reaction. Moreover, surprisingly, thepresence of a luciferin, a molecule which has fluorescent properties andis often present in relatively large quantities in luminescent assays,did not result in significant interference in combinedfluorescent/luminescent assays. Further, surprisingly, two caspases anda luciferase were detected in the same reaction mix, a mix whichincluded a caspase-8 substrate (Z-LETD-aminoluciferin) and two caspase-3substrates, i.e., (Z-DEVD)₂-rhodamine-110 and Ac-DEVD-AMC. The presentinvention thus provides more flexibility in molecules to be employed inmultiplex assays, e.g., substrates for a luminogenic assay incombination with substrates for a fluorogenic assay. Moreover, if twoenzyme-mediated reactions have compatible reagent conditions, the assaycan be a one-step assay.

Accordingly, a combined luminogenic/nonluminogenic assay format of thepresent invention allows multiplexing of assays for one or more peptidesor polypeptides, e.g., enzymes, one or more molecules which are bound byand/or altered by the peptide(s) or polypeptide(s), e.g., a peptide orpolypeptide substrate for each enzyme, and/or one or more cofactors foreach assay, or a combination thereof. Thus, in one embodiment, theinvention provides a method to detect the presence or amount of a firstmolecule for a first enzyme-mediated reaction and the presence or amountof a second molecule for a second enzyme-mediated reaction. The methodincludes contacting a sample suspected of having the first and/or secondmolecules with a reaction mixture for the first and secondenzyme-mediated reactions which lacks the first and/or second molecules.The presence or amount of the first and the second molecules is thendetected. The use of multiplexing which includes a luminescent assayprovides increased sensitivity for the molecule detected using theluminescent assay. Thus, in one embodiment, a reaction mediated by thefirst enzyme yields a luminogenic product, whereas a reaction mediatedby the second enzyme yields a nonluminogenic product. In one embodiment,a combined luminogenic/fluorogenic assay is provided including one inwhich one of the assays provides an internal control. The assaysdescribed herein may be employed with other assays, including reporterassays, nucleic-acid based assays or immunological-based assays andother unrelated enzyme assays.

The invention also provides a method for measuring the activity orpresence of at least one molecule in a sample. The method includesproviding a sample that may contain at least one molecule for anenzyme-mediated reaction, e.g., the sample may contain the enzyme, andcontacting the sample with a reaction mixture for the enzyme-mediatedreaction which lacks the molecule, e.g., the reaction mixture contains asubstrate for the enzyme, so as to yield a reaction mixture wherein thepresence or amount of the molecule is capable of being detected by aluminogenic assay. In one embodiment, the sample and/or reaction mixtureis also contacted with reagents to detect a molecule for a secondenzyme-mediated reaction, where the presence or amount of the moleculefor the second enzyme-mediated reaction is capable of being detected bya nonluminogenic assay.

In one embodiment, the invention provides a method to detect thepresence or amount of a first enzyme and/or a cofactor for a reactionmediated by that enzyme in a sample. The method includes contacting thesample with a first substrate for the first enzyme, a second substratefor a second enzyme, and optionally a third enzyme, to yield a reactionmixture. In one embodiment, at least the first and second enzymes arenot the same, e.g., do not substantially recognize the same substrate,i.e., they do not bind to the same substrate, or if they bind to andreact with the same substrate, one of the enzymes does not react with asubstrate for the other enzyme to the same extent (efficiency), i.e.,one of the enzymes does not react substantially with a substrate for theother enzyme when substrates for both enzymes are present. As usedherein, an enzyme (first enzyme) which does not react substantially witha substrate for a second enzyme includes an enzyme which, in a reactionhaving the second enzyme and equal amounts of a substrate for the firstenzyme and a substrate for the second enzyme, cross reacts with thesubstrate for the second enzyme no more than 25%, e.g., cross reacts15%, 10% or 5% or less, relative to a reaction between the first enzymeand substrate for the first enzyme. The first substrate, a product of areaction between the first substrate and the first enzyme, and/or aproduct of a reaction between the third enzyme and the product of thefirst enzyme and the first substrate, is/are luminogenic. The secondsubstrate, a (second) product of a reaction between the second substrateand the second enzyme, and/or a product of a reaction between anotherenzyme and the second product, is/are not luminogenic but otherwisedetectable. The presence or amount of the first enzyme and/or cofactoris detected or determined. In one embodiment, the presence or amount ofthe second enzyme and/or a cofactor for the reaction mediated by thesecond enzyme is also detected or determined. In one embodiment, atleast the first and second enzymes are not the same. The enzymes to bedetected may be native enzymes or recombinant enzymes, e.g., includingfusion proteins. The optional enzyme(s) added to the sample likewise maybe native or recombinant enzymes.

In another embodiment, the invention provides a method to detect thepresence or amount of a first enzyme and/or a cofactor for a reactionmediated by that enzyme in a sample. The method includes contacting thesample with a first substrate for the first enzyme, a second substratefor a second enzyme, and optionally a third enzyme, to yield a reactionmixture, wherein optionally at least the first and second enzymes arenot the same. The first substrate, a product of a reaction between thefirst substrate and the first enzyme, and/or a product of a reactionbetween the third enzyme and the product of the first enzyme and thefirst substrate, is/are not luminogenic but otherwise detectable. Thesecond substrate, a second product of a reaction between the secondsubstrate and the second enzyme, and/or a product of a reaction betweenanother enzyme and the second product, is/are luminogenic. The presenceor amount of the first enzyme and/or cofactor is detected or determined.In one embodiment, the presence or amount of the second enzyme is alsodetected or determined. The enzymes to be detected or employed in thereaction mixture may be native enzymes or recombinant enzymes.

Further provided is a method of assaying an enzyme-mediated luminescencereaction to detect a first enzyme or cofactor for a reaction mediated bythat enzyme. The method includes contacting a sample with a firstsubstrate for the first enzyme, a second substrate for a second enzyme,and optionally a third enzyme, to yield a reaction mixture, wherein thefirst and second enzymes are not the same. The first substrate, aproduct of the reaction between the first substrate and the firstenzyme, and/or a product of the third enzyme and the product of thefirst enzyme and first substrate, is/are luminogenic. The secondsubstrate, a second product of the reaction between the second substrateand the second enzyme, and/or a product of a reaction between the secondproduct and another enzyme is/are not luminogenic but otherwisedetectable. Luminescence is then detected. The method may furtherinclude detecting the presence or amount of the second enzyme, e.g., bydetecting the presence or amount of the nonluminogenic substrate orproduct(s). In one embodiment, the second enzyme does not bind to orreact with the first substrate, while in another embodiment, the firstenzyme does not bind to or react with the second substrate. In oneembodiment, at least the first and second enzymes are not the same. Theenzymes to be detected or employed in the reaction mixture may be nativeenzymes or recombinant enzymes.

Also provided is a method of assaying an enzyme-mediated luminescencereaction to detect a first enzyme or cofactor for a reaction mediated bythat enzyme. The method includes contacting a sample with a firstsubstrate for the first enzyme, a second substrate for a second enzyme,and a third enzyme, to yield a reaction mixture. The first substrate, aproduct of the reaction between the first substrate and the firstenzyme, and/or a product of the third enzyme and the product of thefirst enzyme and first substrate, is/are not luminogenic but otherwisedetectable. The second substrate, a second product of the reactionbetween the second substrate and the second enzyme, and/or a product ofa reaction between the second product and another enzyme is/areluminogenic. Luminescence is then detected. The method may furtherinclude detecting the presence or amount of the first enzyme or productof the first enzyme and first substrate. In one embodiment, the secondenzyme does not bind to or react substantially with the first substrate,while in another embodiment, the first enzyme does not bind to or reactsubstantially with the second substrate. In one embodiment, at least thefirst and second enzymes are not the same. The enzymes to be detected oremployed in the reaction mixture may be native enzymes or recombinantenzymes, e.g., including fusion proteins.

Further provided is a method to detect the presence or amount of atleast two molecules in a sample. The method includes contacting a samplewith a first substrate for a first enzyme, a second substrate for asecond enzyme, and optionally a third enzyme, to yield a reactionmixture, wherein at least the first and second enzymes are not the same.A reaction between the first enzyme and the first substrate or the thirdenzyme and a product of the reaction between the first substrate and thefirst enzyme yields a luminogenic product. The second substrate, asecond product of the reaction between the second substrate and thesecond enzyme, and/or a product of a reaction between the second productand a different enzyme, is/are not luminogenic. The presence or amountof the first and second enzymes and/or cofactor(s) is then detected. Inone embodiment, luminescence is employed to detect the first enzymeand/or cofactor and fluorescence or colorimetry is employed to detect atleast one other enzyme and/or cofactor. In one embodiment, substratesfor two different enzymes are simultaneously combined with a sample toyield a reaction mixture. A reaction between one of the substrates andone of the enzymes directly or indirectly generates a luminescent signalwhile a reaction between the other substrate and enzyme directly orindirectly generates a fluorescent signal. Following an incubationperiod, the fluorescent signal is employed to detect the presence oramount of one enzyme and/or cofactor and the luminescent signal isemployed to detect the presence or amount of the other enzyme and/orcofactor. Specific buffer conditions can vary with the enzymes and/orcofactor(s) being detected, and can be determined by one of skill in theart of in vitro assays, e.g., enzyme assays. Alternatively, the assaycan be a two-step assay, with reagent adjustment between the first andsecond assays. For example, reagent adjustment can include addition of aquenching agent for the first reaction, and/or an enhancing agent forthe second reaction.

In one embodiment, to detect the first enzyme or cofactor for the firstenzyme-mediated reaction and the second enzyme or cofactor for thesecond enzyme-mediated reaction, the sample is simultaneously contactedwith the first substrate and the second substrate. In anotherembodiment, the sample is contacted with the second substrate before thefirst substrate, or is contacted with the first substrate before thesecond substrate. In one embodiment, the third or different enzyme maybe added with the one or more substrates, before the one or moresubstrates or after the one or more substrates.

In one embodiment, to detect the first substrate or cofactor for thefirst enzyme-mediated reaction and the second substrate or cofactor forthe second enzyme-mediated reaction, the sample is simultaneouslycontacted with the first enzyme and the second enzyme. In anotherembodiment, the sample is contacted with the second enzyme before thefirst enzyme, or is contacted with the first enzyme before the secondenzyme.

In one embodiment, to detect the first enzyme or cofactor for the firstenzyme-mediated reaction and the second substrate or cofactor for thesecond enzyme-mediated reaction, the sample is simultaneously contactedwith the first substrate and the second enzyme. In another embodiment,the sample is contacted with the second enzyme before the firstsubstrate, or is contacted with the first substrate before the secondenzyme. In one embodiment, to detect the first substrate or cofactor forthe first enzyme-mediated reaction and the second enzyme or cofactor forthe second enzyme-mediated reaction, the sample is simultaneouslycontacted with the first enzyme and the second substrate. In anotherembodiment, the sample is contacted with the second substrate before thefirst enzyme, or is contacted with the first enzyme before the secondsubstrate.

The sample employed in the methods of the invention may be a celllysate, an in vitro transcription/translation reaction, a supernatant ofa cell culture, a physiological fluid sample, e.g., a blood, plasma,serum, cerebrospinal fluid, tears or urine sample, and may includeintact cells. The cells, cell lysate, or supernatant may be obtainedfrom prokaryotic cells or eukaryotic cells.

The invention also provides for simultaneous or sequential detection ofthe presence or amount of the first and second proteins, e.g., enzymes,or a cofactor(s) for a reaction mediated by at least one of thoseproteins, e.g., for concurrent reactions or for sequential reactionsoptionally without quenching one of the reactions orenhancing/accelerating one of the reactions. In one embodiment, firstand second substrates are added to the sample simultaneously and theamount or presence of the first enzyme and/or cofactor is detectedbefore the amount or presence of the second enzyme and/or cofactor isdetected. In another embodiment, the first and second substrates areadded to the sample simultaneously and the presence or amount of thesecond enzyme and/or cofactor is detected before the amount or presenceof the first enzyme and/or cofactor is detected. Alternatively, thefirst and second substrates are added to the sample simultaneously andthe presence or amount of the first and second enzymes and/or cofactorsis detected simultaneously. Preferably, the presence or amount ofenzymes and/or cofactors are detected in a single reaction, e.g., allreactions are conducted in a single receptacle, e.g., well.

In another embodiment, the invention provides a method to detect thepresence or amount of a molecule for an enzyme-mediated reaction inconjunction with expression of a fluorescent protein, e.g., greenfluorescent protein. For example, cells which transiently or stablyexpress a fluorescent protein, or a protein that can be labeled in cellsto become fluorescent, such as dehalogenase, can be assayed for thepresence or amount of the fluorescent protein via a fluorogenic assay aswell as assayed for at least one additional molecule, e.g., an enzyme,substrate or co-factor for a reaction mediated by the enzyme, whichmolecule is present in or secreted by the cells via a luminogenic assay.In one embodiment, the presence or amount of a different molecule isalso detected or determined, for example, in a nonluminogenic assay. Thepresence or amount of the molecule(s) may then be normalized using datagenerated from the fluorescent protein.

Thus, the invention provides a method to detect the presence or amountof a molecule for a reaction mediated by a first enzyme. The methodincludes contacting a sample which comprises cells which express afluorescent protein with a reaction mixture for the first enzyme whichlacks the molecule, and optionally a second enzyme. A reaction mediatedby the first enzyme yields a luminogenic product. The presence or amountof the molecule and the presence or amount of the fluorescent proteinare then detected.

In one embodiment, for luminogenic and/or fluorogenic assays which yieldproducts with different characteristics, e.g., different colors, furthermultiplexing (i.e., with other substrates) may be employed. For example,further multiplexing may include using different colors emitted bydifferent luciferase based reactions or substrates or a fluorogenicassay with different excitation/emission spectra.

Also provided are kits which include one or more reagents for use in theassays of the invention.

The assay also has use as a drug discovery tool. Many drug-testingcompounds have fluorescent properties that may interfere with afluorescent/luminescent multiplex assay. The present invention providesassays to detect false results. As described herein, the same consensussubstrate sequence for caspase-3 was linked to different reportermolecules with distinct spectral readouts, e.g., two with a fluorescentreadout and one with a luminescent readout. Caspase-3 and luciferasewere assayed in the presence and the absence of a caspase-3 inhibitor ora luciferase inhibitor. The data showed that there was very littleinterference between the three reporter molecules, and that luciferasecould be used in a normalizing assay to control for false results.

Thus, the presence or amount of a modulator, for instance, an inhibitor,of an enzyme may be detected using a multiplex assay of the invention,e.g., a combined fluorogenic/luminogenic assay. In one embodiment, themethod includes providing a reaction mixture comprising a nonluminogenicsubstrate for a first enzyme, a second substrate for the first enzyme, asecond enzyme for a luminogenic assay, and a test agent. A reactionbetween the nonluminogenic substrate and the first enzyme but not thesecond substrate and the first enzyme yields a nonluminogenic product,and a reaction between the second substrate and the first enzyme yieldsa substrate for the second enzyme, e.g., a substrate for a luciferase. Areaction between the substrate for the second enzyme and the secondenzyme yields a luminogenic product. The presence or amount of theluminogenic product and the nonluminogenic product is compared in testand control reactions. Comparison of the two results indicates theeffect of the modulator on the enzyme for the luminogenic assay, whichcan eliminate false results.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-B. Multiplex assay measuring the enzyme activities of caspase-3and caspase-8 in the presence of both luminogenic and fluorogenic assayreagents. A) Relative light units (RLU) versus time. B) Relativefluorescence units (RFU) over time.

FIGS. 2A-C. Multiplex assay of caspase-3 and caspase-8. A) Signal tobackground fluorescence for AMC. B) Signal to background fluorescencefor rhodamine-110. C) Signal to background luminescence.

FIGS. 3A-C. Triplex assay measuring the activities of caspase-3,caspase-8, and trypsin. A) RFU for rhodamine-110; B) RFU for AMC; C)RLU.

FIGS. 4A-D. Multiplex assay measuring a protease (caspase-3) and anon-protease (β-galactosidase) enzyme. A) and C), RLU at ½ hour and 18hours, respectively. B) and D) RFU at 2 hours and 18 hours,respectively.

FIGS. 5A-C. Excitation and emission spectra of luciferin (A),aminoluciferin (B) and Z-LETD-aminoluciferin (C).

FIG. 6. Signal to background ratios in three channels; rhodamine-110,AMC and luminescence, in a caspase-3 assay.

FIGS. 7A-D. Multiplex fluorogenic and luminogenic assays measuringlactate dehydrogenase (LDH) activity and adenosine triphosphate (ATP).A) and C) RLU versus ATP concentration. B) and D) RFU versus LDHdilution.

FIGS. 8A-D. Multiplex fluorogenic and luminogenic assays measuring LDHand caspase-3. A) and C) RLU versus caspase-3 concentration. B) and D)RFU versus LDH dilution.

FIGS. 9A-D. Multiplex fluorogenic and luminogenic assays measuringprotein kinase A (PKA) and caspase-3. A) and C) RLU versus caspase-3concentration. B) and D) RFU versus PKA concentration.

FIG. 10. Multiplex fluorogenic and luminogenic assays measuringcaspase-3 and Renilla luciferase (luc). A) and C) RLU versusstaurosporine concentration. B) and D) RFU versus caspase-3concentration.

DETAILED DESCRIPTION OF THE INVENTION

The invention provides a multiplexed assay method in which at least twodifferent molecules which bind to and/or are altered by a protein (e.g.,peptide or polypeptide) are provided either simultaneously orsequentially in a reaction mixture to detect one or more moietiesincluding proteins (peptides or polypeptides), e.g., enzymes, orsubstrates or cofactors for reactions. For instance, one or moreenzyme-mediated reactions are performed under conditions effective toconvert at least one enzyme substrate to a product of a reaction betweenthe substrate and the enzyme. Preferably, each molecule in the reactionmixture, e.g., substrate, or product in the reaction has a differentcharacteristic from other molecule(s) or product(s), and, in oneembodiment, at least one molecule includes a reporter molecule capableof directly or indirectly producing a detectable signal. The resultingsignal is related to the presence or amount of the molecule to bedetected. In one embodiment, the method includes performing two or moreenzyme reactions in the presence of at least two different enzymesubstrates under conditions effective to convert each substrate to acorresponding product, where at least the substrate or product of eachreaction, and/or a product of a reaction between one of the products anda third, e.g., different, enzyme, has a different detectablecharacteristic, e.g., a different optical characteristic, from the othersubstrate(s) and/or product(s). After performing the reactions, eithersimultaneously or sequentially, the presence or amount of one or moresubstrates or one or more products of the reaction(s) is/are detected ordetermined. From this, the presence or amount of the correspondingenzyme(s) and/or cofactors can be determined.

Thus, two general types of multiplexed assays are contemplated. In thefirst, multiple moieties, e.g., one or more enzymes, one or moresubstrates and/or one or more cofactors for an enzyme-mediated reaction,are assayed in the same reaction mixture. Each enzyme is capable ofconverting at least one of the substrates to a corresponding product,where the substrate(s) and/or corresponding product(s), or product(s) ofa reaction between one of the corresponding products and another enzyme,have different detectable characteristics that allow the substratesand/or the products to be individually detected when present in the samereaction mixture. The order of adding the molecules for the assays ofthe present invention can vary. Thus, individual reactions may beinitiated and/or conducted simultaneously or sequentially. If initiatedand conducted sequentially, the different detectable characteristics mayrequire different detection methods, and/or adjustments to reactionconditions, e.g., reagent concentration, temperatures or additionalreagents, may be performed. For instance, a quenching agent or enhancingagent may be added between reactions (see, e.g., U.S. Pat. Nos.5,774,320 and 6,586,196, the disclosures of which are specificallyincorporated by reference herein). In one preferred embodiment, the twoor more reactions are carried out simultaneously in a single reactionmixture, where each of the enzymes is effective to convert one of thesubstrates in the reaction mixture to a product. This embodiment may beused, for example, to determine the presence or amount of at least twodifferent enzymes and/or cofactors in a cell, cell lysate or cellsupernatant. In addition, the reaction may contain one or more testagents, e.g., enzyme inhibitors or activators, and/or differentconcentrations of inhibitors, activators, or substrates.

Optionally, the assays are employed as a homogeneous assay, e.g., theone or more substrates and additional components are mixed prior toadding the mixture to the sample. Results may be read without additionaltransfer of reagents.

In a second assay type, two or more enzyme-mediated reactions arecarried out in tandem. The separate reactions may be performed at thesame time or at different times. The reactions may contain one or moreof the same or different enzymes, one or more of the same or differenttest agents, e.g., enzyme inhibitors or activators, and/or differentconcentrations of inhibitors, activators, or substrates. In oneembodiment, each reaction mixture contains at least two substratescapable of being converted to a product, where the substrate(s) and/orcorresponding product(s), and/or a product(s) of a reaction between theproduct of one of the enzyme/substrate pairs and a different enzyme,have different detectable characteristics.

The assays of the present invention thus allow the detection of multipleenzymes or cofactors in a sample, e.g., a sample which includeseukaryotic cells, e.g., yeast, avian, plant, insect or mammalian cells,including but not limited to human, simian, murine, canine, bovine,equine, feline, ovine, caprine or swine cells, or prokaryotic cells, orcells from two or more different organisms, or cell lysates orsupernatants thereof. The cells may not have been genetically modifiedvia recombinant techniques (nonrecombinant cells), or may be recombinantcells which are transiently transfected with recombinant DNA and/or thegenome of which is stably augmented with a recombinant DNA, or whichgenome has been modified to disrupt a gene, e.g., disrupt a promoter,intron or open reading frame, or replace one DNA fragment with another.The recombinant DNA or replacement DNA fragment may encode a molecule tobe detected by the methods of the invention, a moiety which alters thelevel or activity of the molecule to be detected, and/or a gene productunrelated to the molecule or moiety that alters the level or activity ofthe molecule.

In one embodiment, the methods according to the present inventionprovide a rapid, highly sensitive method for simultaneously orsequentially detecting multiple moieties including enzymes in a singlesample such as an aliquot of cells or a lysate thereof. In oneembodiment, the method includes quantifying the presence or amount(activity) of a first enzyme, substrate or cofactor in a luminogenicassay and quantifying the presence or amount of a second enzyme,substrate or cofactor in a nonluminogenic assay, such as a fluorogenicassay. In one embodiment, reagents, e.g., substrates, for each reactionmay be added together or sequentially. In another embodiment, the methodincludes quantifying the presence or amount of a first enzyme, substrateor cofactor in a fluorogenic assay and quantifying the presence oramount of a second enzyme, substrate or cofactor in a luminogenic assay.Thus, in another embodiment, the method includes quantifying thepresence or amount of a cofactor in a luminogenic assay and quantifyinga different molecule in a nonluminogenic assay. In yet anotherembodiment, the method includes quantifying the presence or amount of acofactor in a nonluminogenic assay and quantifying a different moleculein a luminogenic assay. The intensity of the luminogenic ornonluminogenic signal is a function of the presence or amount of therespective molecule.

In one embodiment, the present invention relates to a method ofmeasuring the presence or amount of multiple enzymes in a single aliquotof cells or a lysate thereof. In one embodiment, at least one of theenzymes is an endogenous enzyme, For example, in one embodiment, thepresent invention provides an improved, sensitive method for monitoringthe activity of at least one protease and optionally another enzyme inpreparations comprising the protease and the other enzyme, includingpurified preparations from either prokaryotic or eukaryotic cells, celllysates or supernatants of cells such as cultured eukaryotic cells,e.g., mammalian cells. For enzymes present in different cellularlocations, such as a secreted and an intracellular protease, a substratefor each enzyme can be added to a well with intact cells. The presenceor amount of the secreted protease may be detected prior to detection ofthe intracellular protease, such as after cell lysis, e.g., where thedetection of the intracellular protease is in the same receptacle, forinstance, same well, as that for the secreted protease. In oneembodiment, a non-cell permeant substrate for an intracellular proteaseand a substrate for a secreted protease are added to a sample comprisingcells and the cells are then lysed. Detection of the secreted proteasemay be before cell lysis or after cell lysis. In another embodiment, anon-cell permeant substrate for an intracellular enzyme or a secretedprotease, and a cell permanent substrate for a second intracellularenzyme are added to a sample comprising cells. The presence of thesecond intracellular enzyme and the secreted protease may be detectedwithout lysis. In yet another embodiment, a triplex assay is performedto detect a secreted protease, an intracellular enzyme (by employingeither a cell permeant substrate or non-cell permeant substrate) and asecond intracellular enzyme (by employing either a cell permeantsubstrate or a non-cell permeant substrate). In one embodiment, thesecreted protein is detected using fluorescence or spectrophotometry.

The present methods can be employed to detect any molecule including anyenzyme or any set of enzymes. The enzymes employed in the methods,either enzymes to be detected or enzymes which are useful to detect asubstrate or cofactor, can be selected from any combination of enzymesincluding recombinant and endogenous (native) enzymes. In oneembodiment, all of the enzymes to be detected are endogenous enzymes. Inanother embodiment, two enzymes to be detected are endogenous enzymesand another enzyme is a recombinant enzyme. In another embodiment, oneenzyme is an endogenous enzyme and another enzyme is a recombinantenzyme. Other combinations apparent to one of ordinary skill in the artcan be used in the present assays and methods according to the teachingsherein. The enzymes include but are not limited to proteases,phosphatases, peroxidases, sulfatases, peptidases, and glycosidases. Theenzymes may be from different groups based on the nature of thecatalyzed reaction, groups including but not limited to hydrolases,oxidoreductases, lyases, transferases, isomerases, ligases, orsynthases, or they may be from the same group so long as at least one ofthe enzymes has a partially overlapping or preferably a substantiallydifferent substrate specificity relative to at least one of the otherenzymes. Of particular interest are classes of enzymes that havephysiological significance. These enzymes include protein kinases,peptidases, esterases, protein phosphatases, isomerases, glycosylases,synthetases, proteases, dehydrogenases, oxidases, reductases, methylasesand the like. Enzymes of interest include those involved in making orhydrolyzing esters, both organic and inorganic, glycosylating, andhydrolyzing amides. In any class, there may be further subdivisions, asin the kinases, where the kinase may be specific for phosphorylation ofserine, threonine and/or tyrosine residues in peptides and proteins.Thus, the enzymes may be, for example, kinases from different functionalgroups of kinases, including cyclic nucleotide-regulated proteinkinases, protein kinase C, kinases regulated by Ca²⁺/CaM,cyclin-dependent kinases, ERK/MAP kinases, and protein-tyrosine kinases.The kinase may be a protein kinase enzyme in a signaling pathway,effective to phosphorylate an oligopeptide substrate, such as ERKkinase, S6 kinase, IR kinase, P38 kinase, and AbI kinase. For these, thesubstrates can include an oligopeptide substrate. Other kinases ofinterest may include, for example, Src kinase, JNK, MAP kinase,cyclin-dependent kinases, P53 kinases, platelet-derived growth factorreceptor, epidermal growth factor receptor, and MEK.

In particular, enzymes that are useful in the present invention includeany protein that exhibits enzymatic activity, e.g., lipases,phospholipases, sulphatases, ureases, peptidases, proteases andesterases, including acid phosphatases, glucosidases, glucuronidases,galactosidases, carboxylesterases, and luciferases. In one embodiment,one of the enzymes is a hydrolytic enzyme. In another embodiment, atleast two of the enzymes are hydrolytic enzymes. Examples of hydrolyticenzymes include alkaline and acid phosphatases, esterases,decarboxylases, phospholipase D, P-xylosidase, β-D-fucosidase,thioglucosidase, β-D-galactosidase, α-D-galactosidase, α-D-glucosidase,β-D-glucosidase, β-D-glucuronidase, α-D-mannosidase, β-D-mannosidase,β-D-fructofuranosidase, and β-D-glucosiduronase.

A substrate or cofactor for any particular enzyme-mediated reaction isknown to those of skill in the art. Exemplary cleavage sites for someproteases are set forth in Table 1.

TABLE 1 Protease Cut Site(s) Aminopeptidase M Hydrolysis from freeN-terminus Carboxypeptidase Y Hydrolysis from C-terminus Caspase-1,4,5W/LEHD-X Caspase-2,3,7 DEXD-X Caspase-6,8,9 L/VEXD-X Chymotrypsin Y-X,F-X, T-X, (L-X, M-X, A-X, E-X) Factor Xa IEGR-X Pepsin F-Z, M-Z, L-Z,W-Z (where Z is a hydrophobic residue) but will cleave others TEVE(N)XYXQ-S/G Thrombin R-X Trypsin R-X, K-X Tryptase PRNK-X β-secretaseEISEVK/NM/L-DAEFRHD, e.g., SEVNL-DAEFR X is one or more amino acids

For alkaline phosphatase, it is preferable that the substrate includes aphosphate-containing dioxetane, such as3-(2′-spiroadamantane)-4-methoxy-4-(3″-phosphoryloxy)phenyl-1,2-dioxetane,disodium salt, or disodium3-(4-methoxyspiro[1,2-dioxetane-3,2′(5′-chloro)-tricyclo-[3.3.1.1^(3,7)]decan]-4-yl]phenylphosphate, or disodium2-chloro-5-(4-methoxyspiro{1,2-dioxetane-3,2′-(⁵′-chloro)-tricyclo{3.3.1.13,7]decan}-4-yl)-1-phenyl phosphate or disodium2-chloro-5-(⁴-methoxyspiro{1,2-dioxetane-3,2′-tricyclo[3.3.1.13,7]decan}-4-yl)-1-phenzylphosphate (AMPPD, CSPD, CDP-Star® and ADP-Star™, respectively).

For β-galactosidase, the substrate preferably includes a dioxetanecontaining galactosidase-cleavable or galactopyranoside groups. Theluminescence in the assay results from the enzymatic cleavage of thesugar moiety from the dioxetane substrate. Examples of such substratesinclude3-(2′-spiroadamantane)-4-methoxy-4-(3″-β-D-galactopyranosyl)phenyl-1,2-dioxetane(AMPGD),3-(4-methoxyspiro[1,2-dioxetane-3,2′-(5′-chloro)tricyclo[3.3.1.1^(3,7)]-decan]-4-yl-phenyl-β-D-galactopyranoside(Galacton®),5-chloro-3-(methoxyspiro[1,2-dioxetane-3,2′-(5′-chloro)tricyclo[3.3.1^(3,7)]decan-4-yl-phenyl-β-D-galactopyranoside(Galacton-Plus®), and2-chloro-5-(4-methoxyspiro[1,2-dioxetane-3,2′(5′-chloro)-tricyclo-[3.3.1.1^(3,7)]decan]-4-yl)phenylβ-D-galactopyranoside (Galacton-Star®).

In assays for β-glucuronidase and β-glucosidase, the substrate includesa dioxetane containing β-glucuronidase-cleavable groups such as aglucuronide, e.g., sodium 3-(4-methoxyspiro{1,2-dioxetane-3,2′-(5′-chloro)-tricyclo[3.3.1.1^(3,7)]decan}-4-yl)phenyl-β-D-glucuronate(Glucuron™). In assays for a carboxyl esterase, the substrate includes asuitable ester group bound to the dioxetane. In assays for proteases andphospholipases, the substrate includes a suitable enzyme-cleavable groupbound to the dioxetane.

Preferably, the substrates for each enzyme in the assay are different.For assays which include one dioxetane containing substrate, thesubstrate optionally contains a substituted or unsubstituted adamantylgroup, a Y group which may be substituted or unsubstituted and an enzymecleavable group. Examples of preferred dioxetanes include thosementioned above, e.g., those referred to as Galacton®, Galacton-Plus®,CDP-Star®, Glucuron™, AMPPD, Galacton-Star®, and ADP-Star™, as well as3-(4-methoxyspiro{1,2-dioxetane-3,2′-(5′-chloro)-tricyclo[3.3.1.1^(3,7)]decan}-4-yl)phenyl-β-D-glucopyranoside(Glucon™), CSPD, disodium3-chloro-5-(4-methoxyspiro{1,2-dioxetane-3,2′(5′-chloro)-tricyclo-[3.3.1.1^(3,7)]decan)-4-yl)-1-phenylphosphate (CDP).

Preferably, a substrate for at least one enzyme to be detected ismodified to contain a reporter molecule. A reporter molecule is anymolecule that allows a substrate linked to that molecule, a productresulting from a reaction between the enzyme and the substrate, or aproduct of a reaction between that product and another enzyme, to bedifferentially detected, preferably quantitatively. Reporter moleculesinclude but are not limited to optic molecules such as fluorophores, anabsorptive colored particle or a dye, radiolabels, enzymes such as acatalytic moiety that is effective to catalyze a detectable reaction inthe presence of suitable reaction components, a subunit or fragment ofan enzyme that is functional when associated with other subunit(s) orfragment(s), or a substrate for a subsequent reaction, e.g., one inwhich the product of that reaction is detectable. As used herein, a“fluorophore” includes a molecule which is capable of absorbing energyat a wavelength range and releasing energy at a wavelength range otherthan the absorbance range. The term “excitation wavelength” refers tothe range of wavelengths at which a fluorophore absorbs energy. The term“emission wavelength” refers to the range of wavelengths that thefluorophore releases energy or fluoresces.

In one embodiment, the reporter molecule fluoresces. One group offluorescers is the xanthene dyes, which include the fluoresceins,rosamines and rhodamines. These compounds are commercially availablewith substituents on the phenyl group, which can be used as the site forbonding or as the bonding functionality. For example, amino andisothiocyanate substituted fluorescein compounds are available.

Another group of fluorescent compounds are the naphthylamines, having anamino group in the alpha or beta position, usually alpha position.Included among the naphthylamino compounds are1-dimethylaminonaphthyl-5-sulfonate, 1-anilino-8-napththalene sulfonateand 2-p-toluidinyl-6-naphthalene sulfonate. Some naphthalene compoundsare found to have some non-specific binding to protein, so that theiruse requires employing an assay medium where the amount of protein isminimized. Other fluorescers are multidentate ligands that includenitrogen-containing macrocycles, which have conjugated ring systems withpi-electrons. These macrocycles may be optionally substituted, includingsubstitution on bridging carbons or on nitrogens. Suitable macrocyclesinclude derivatives of porphyrins, azaporphyrins, corrins, sapphyrinsand porphycenes and other like macrocycles, which contain electrons thatare extensively delocalized. The azaporphyrin derivatives includephthalocyanine, benzotriazaporphyrin and naphthalocyanine and theirderivatives.

In some instances fluorescent fusion proteins may be employed, e.g., agreen, red or blue fluorescent protein or other fluorescent proteinfused to a polypeptide substrate. In other embodiments, a fluorescentprotein may itself be a substrate for a hydrolytic enzyme. A“fluorescent protein” is a full-length fluorescent protein or afluorescent fragment thereof.

A non-limiting list of chemical fluorophores of use in the invention,along with their excitation and emission wavelengths, is shown in Table2. Excitation and emission values can change depending on reactionconditions, such as pH, buffer system, or solvent.

TABLE 2 Fluorophore Excitation (nm) Emission (nm) Fluorescein (FITC) 495525 Hoechst 33258 360 470 R-Phycoerythrin (PE) 488 578 Rhodamine (TRITC)552 570 Quantum Red ™ 488 670 Texas Red 596 620 Cy3 552 570Rhodamine-110 499 521 AFC 380 500 AMC 342 441 Resorufin 571 585 BODIPYFL 504 512 BODIPY TR 591 620

In one embodiment, one of the enzymes is detected using a substratewhich includes an amino-modified luciferin or a carboxy protectedderivative thereof, which modification includes a substrate for theenzyme. In one embodiment, the modification is one or more amino acidresidues which include a recognition site for a protease. In oneembodiment, the substrate is covalently linked to the amino group ofaminoluciferin or a carboxy-modified derivative thereof via a peptidebond. Preferably, the N-terminus of a peptide or protein substrate ismodified to prevent degradation by aminopeptidases, e.g., using anamino-terminal protecting group. In the absence of the appropriateenzyme or cofactor, a mixture including such a substrate and luciferasegenerates minimal light as minimal aminoluciferin is present. In thepresence of the appropriate enzyme, the bond linking the substrate andaminoluciferin can be cleaved by the enzyme to yield aminoluciferin, asubstrate for luciferase. Thus, in the presence of luciferase, forinstance, a native, recombinant or mutant luciferase, and any cofactorsand appropriate reaction conditions, light is generated, which isproportional to the presence or activity of the enzyme.

In one embodiment, one of the enzymes is detected using a substratewhich includes a fluorophore. In one embodiment, the substrate includesone or more amino acid residues which include a recognition site for aprotease. In one embodiment, the substrate is covalently linked to oneor more fluorophores. In the absence of the appropriate enzyme orcofactor, a mixture including such a substrate generates minimal lightat the emission wavelength as the fluorescent properties of thefluorophore are quenched, e.g., by the proximity of the quenching groupsuch that the properties of a substrate-fluorophore conjugate arechanged, resulting in altered, e.g., reduced, fluorescent properties forthe conjugate relative to the fluorophore alone. In the presence of theappropriate enzyme, cleavage of the conjugate yields the fluorophore. Inanother embodiment, prior to cleavage, the conjugate is fluorescent butafter cleavage with the enzyme, the product(s) have altered spectra.

In one embodiment, the conditions for at least two of the reactions arecompatible. For instance, the conditions for at least 2 enzymes, andpreferably the conditions for 3 or more enzymes, e.g., 4 or moreenzymes, are compatible. A group of similar enzymes will generally havecompatible reaction conditions, such as pH and ionic strength, however,cofactor requirements, metal ion requirements, and the like, involvingassay components having relatively low mass concentrations, e.g.,cofactors, need not be common. Common conditions include conditions suchthat each of the enzymes provides a measurable rate during the course ofthe reaction and will generally be that each of the enzymes has at leastabout 10%, usually at least about 20%, preferably at least about 50%, ofits maximum turnover rate for the particular substrate, withoutsignificant interference from the components added for the otherenzyme(s).

Alternatively, the conditions for one reaction may not be compatiblewith another reaction although substrates for both reactions arepresent. In such embodiments, one enzyme is active but cannot react withits substrate. In one embodiment, for example, where conditions for tworeactions are not compatible, individual enzyme-assay reactions arecarried out sequentially and/or in separate reaction mixtures. Followingthe enzyme assay, the reaction mixture (or a portion thereof) may becombined with another reaction. Each individual reaction mixture maycontain one or more enzymes and one or more substrates. In its simplestform, a single enzyme to be assayed and a single substrate for thatenzyme are in each reaction mixture. The set of substrates employed inthe reaction has the same general properties as that required in thesingle-reaction multiplexed assay. That is, each substrate and/orcorresponding product have unique characteristics, allowing them to bedistinguished from one another.

The order of detection of molecules in the reactions can vary. In oneembodiment, regardless of whether reactions are initiated at the sametime or not, the molecule detected by a luminogenic assay is detected,then the molecule detected by the nonluminogenic assay is detected.Alternatively, regardless of whether reactions are initiated at the sametime or not, the molecule detected by the nonluminogenic assay isdetected, then the molecule detected by the luminogenic assay isdetected. In other embodiments, the presence or amount of two or moremolecules is detected essentially simultaneously. In one embodiment, thepresence or activity of one molecule to be detected is substantiallydecreased prior to detecting the presence or activity of the secondmolecule, e.g., by waiting until the first signal has diminished, e.g.,by at least 50%, or by adding a quenching agent for the first reaction.Thus, in some embodiments, one or more of the reactions are terminated,e.g., by inhibiting an enzyme for the reaction, prior to detection.Preferably, the signal produced by one assay does not substantiallyinterfere with the quantification of the signal produced by at least oneother assay.

The present invention also provides kits for detecting the presence oractivity of one or more peptides or proteins, molecules which bind toand/or are altered by the peptides or proteins, or cofactors in a samplesuch as a sample including intact cells, a cell lysate, e.g., a lysatewhich is at least partially purified, or a cellular supernatant. Such akit includes at least one reagent for quantifying at least one of thepeptides and/or proteins, molecules bound by and/or altered by thepeptides and/or proteins, or cofactors, such as a substrate for at leastone enzyme.

The invention will be further described by the following non-limitingexamples. For all examples, suitable control reactions are readilydesigned by those skilled in the art.

EXAMPLE I Fluorescent/Luminescent Multiplex Assays A. Measurement ofCaspase-3 and Caspase-8 in a Single Well, Multiplex Assay

Caspase-Glo™ 8 Reagent (Caspase-Glo™ 8 Assay System, Promega, Corp.) wasevaluated for its ability to allow multiplexing of homogeneousluminogenic caspase-8 and nonluminogenic caspase-3 enzyme assays.Caspase-Glo™ 8 Reagent is comprised of Caspase-Glo™ 8 Buffer and theluminogenic substrate Z-LETD-aminoluciferin. For the luminogenic assaysin FIG. 1A, either Caspase-Glo™ 8 Reagent (diamonds) or Caspase-Glo™ 8Reagent also containing 50 μM of the fluorogenic substrate forcaspase-3, (Z-DEVD)₂-rhodamine-110 (squares), was used to demonstratethe feasibility of a multiplexed luminogenic and nonluminogenic assay.For the fluorogenic assay in FIG. 1B, Caspase-Glo™ 8 Buffer containingeither 50 μM (Z-DEVD)₂-rhodamine-110 and 10 mM DTT (diamonds) or 50 μM(Z-DEVD)₂-rhodamine-110 and Z-LETD-aminoluciferin (squares) were used.

Dilutions of caspase-8 enzyme, caspase-3 enzyme, and combined caspase-8and caspase-3 enzymes (Biomol Research Laboratories) were prepared inRPMI 1640 (Sigma Corporation) to a final concentration of 100 units/ml.100 μl of caspase-8 dilutions, a mixture of caspase-8 and caspase-3dilutions, or caspase-3 dilutions, were added to separate wells of a96-well plate. 100 μl of Caspase-Glo™ 8 Reagent with or without 50 μM(Z-DEVD)₂-rhodamine-110 (FIG. 1A), or 100 μl of Caspase-Glo™ 8 Buffersupplemented with (Z-DEVD)₂-rhodamine 110 and DTT with or withoutZ-LETD-aminoluciferin (FIG. 1B) were added to reach a final volume of200 μl/well. The reaction plate was incubated at room temperature for atleast ten minutes on a plate shaker.

After incubation, relative luminescence was determined using a DYNEXLaboratories MLX™ plate luminometer, and relative fluorescence wasmeasured with a CytoFluor II Fluorescent plate reader outfitted with a485_(EX)/530_(EM) filter set.

Results

The simultaneous measurement of fluorescence and luminescence for twoprotease enzymes in a single well is shown in FIG. 1. As seen in FIG.1A, the presence of caspase-3 and its fluorogenic substrate,(Z-DEVD)₂-rhodamine-110, in a luminogenic assay for caspase-8 (squares)does not greatly alter the luminescent reaction. Similarly, as seen inFIG. 1B, the presence of caspase-8 and its luminogenic substrateZ-LETD-aminoluciferin in a fluorogenic assay for caspase-3 (squares)does not impact the fluorogenic assay for caspase-3.

B. Background Determinations for a Caspase-3 and Caspase-8 MultiplexAssay

Various concentrations of luminogenic and fluorogenic reagents,including caspase enzymes and substrates thereof, and buffer componentswere combined to establish each constituent's contribution tofluorescence and/or luminescence. The fluorogenic substrate(Z-DEVD)₂-rhodamine-110 reports caspase-3 activity in the rhodaminechannel (485_(EX)/520_(EM)) and the fluorogenic substrate Ac-DEVD-AMCreports the caspase-3 activity in the AMC channel (360_(EX)/460_(EM)),while the substrate Z-LETD-aminoluciferin reports caspase-8 activityduring luminescence measurement. Table III describes the amount of eachcomponent (μl) for twelve different reaction conditions resulting in atotal volume of around 500 μl of master mix, or 100 μl of mastermix/reaction (n=4) for each reaction condition. For the ‘caspase added’row, the number in this row defines the type of caspase added inoverabundance and does not describe a volume.

TABLE III Component 1 2 3 4 5 6 7 8 9 10 11 12 Caspase-Glo ™ 8 Buffer400 400 400 400 400 400 400 400 400 400 400 495 Caspase-Glo ™ 8lyophilized 100 na na 100 100 100 100 100 100 100 100 na substratereconstituted in 1 ml of water 5 mM (Z-DEVD)₂-rhodamine- na 5 na 5 5 nana na 5 5 na 5 110 5 mM Ac-DEVD-AMC na na 5 Na na 5 na na na na na naDMSO 5 na na Na na na 5 5 na na 5 na 100 mM Hepes na 80 80 Na na na nana na na na na 1 M DTT na 20 20 Na na na na na na na na na Caspase-3inhibitor (in excess) na na na Na Yes na na Yes na na Yes na Caspaseadded 8 3 3 8&3 8&3 8&3 3 3 8 3 8 3 1. Caspase-8 luc control 2.Caspase-3 rhodamine-110 control 3. Caspase-3 AMC control 4. Multiplexcontrol with rhodamine-110 5. Multiplex control with rhodamine-110 +inhibitor 6. Multiplex control with AMC 7. Capase-3 with aminoluciferinmix 8. Caspase-3 with aminoluciferin mix + inhibitor 9. Caspase-8 withrhodamine-110 10. Caspase-3 with aminoluciferin mix 11. Caspase-8 withaminoluciferin mix + inhibitor 12. Caspase-3 with rhodamine-110 withoutaminoluciferin mix

The components from Table III were added to replicate wells andreactions were incubated at room temperature for two hours. The bufferemployed was that from the Caspase-Glo™ 8 Assay System. DMSO wasobtained from Sigma-Aldrich and the DTT was obtained from Amresco. Thesubstrates and inhibitors were obtained from Promega Corp.

Relative luminescence was determined using a DYNEX Laboratories MLX™plate luminometer. Fluorescence was determined using a CytoFluor IIFluorescent plate reader outfitted with a 485_(EX)/530_(EM) filter setfor rhodamine-110 and then 360_(EX)/460_(EM) for the AMC channel.

Results

For FIGS. 2A, B, and C, all carats represent where either fluorescenceor luminescence indicating enzyme activity was expected. FIG. 2A showsthe signal for AMC fluorescence in each reaction. Fluorescence abovebackground was only present where the appropriate substrate/enzymecombination of Ac-DEVD-AMC and caspase-3 was present (reactionconditions 3 and 6). FIG. 2B shows the signal for rhodamine-110fluorescence in each reaction. Fluorescence above background was presentwhere the substrate/enzyme combination of(Z-DEVD)₂-rhodamine-110/caspase-3 was present (reaction conditions 2, 4,10, and 12), except when a caspase-3 inhibitor was present (reactioncondition 5). For luminescence signal above background (FIG. 2C), thosereactions with the appropriate substrate/enzyme combination ofZ-LETD-aminoluciferin/caspase-8 showed signal above background (reactionconditions 1, 4, 6, 7, 9, and 10), except those reaction conditionswhere a caspase inhibitor was present (reaction conditions 5, 8, and11). The data thus demonstrate that there was negligible contribution ofreaction components to background fluorescence and luminescencemeasurements under these conditions.

C. Measurement of Caspase-3, Caspase-8, and Trypsin in a Single Well,Triplex Assay

Dilutions of detectable levels of caspase-8 (150 units/ml, BiomolResearch Laboratories), caspase-3 (Pharmingen Corp.), trypsin (SigmaCorp.), and a combination of all three enzymes, were prepared inDulbecco's phosphate buffered saline (Sigma Corp.). 100 μl of eachenzyme dilution were added to the wells of a 96-well plate and 100 μl ofeach substrate, either singly or in combination as appropriate, wereadded to the corresponding wells: substrate (Z-DEVD)₂-rhodamine-110 forcaspase-3, substrate Z-PRNK-AMC for trypsin (as described in U.S. patentapplication Ser. No. 09/955,639 as a substrate for beta-tryptases butwith a recognized lesser utility for trypsin, incorporated herein in itsentirety), and substrate Z-LETD-aminoluciferin for caspase-8. WhenCaspase-Glo™ 8 Buffer was employed with a substrate for a fluorogenicassay, 10 mM DTT was included. Plates were incubated for at least tenminutes at room temperature on a plate shaker.

Following incubation, relative luminescence for caspase-8 activity wasmeasured using BMG Fluorostar (BMG Labtechnologies Ltd.). Relativefluorescence was determined using the Labsystems Fluoroskan Ascent platereader. For caspase-3 activity, a filter set of 485_(EX)/527_(EM) wasutilized. For trypsin activity, a filter set of 360_(EX)/460_(EM) wasused.

Results

As shown in FIG. 3A, the conditions employed to detect caspase-3 in areaction with three different substrates and corresponding enzymescombined (the triplex assay) yielded relatively high fluorescence overthat of the control conditions. When comparing the activity of caspase-3in the triplex assay (all substrates with all enzymes) to that ofcaspase-3 alone, caspase-3 activity was greater than background whencaspase-3 was in the same reaction with the other triplex enzymereactions. Similar results were seen for trypsin (FIG. 3B) and caspase-8(FIG. 3C), albeit not to the same extent as with caspase-3.

D. Measurement of Caspase-3 and β-Galactosidase in a Single Well,Multiplex Format

Reagents were prepared by reconstituting Beta-Glo® lyophilized substratewith Beta-Glo® Buffer (Beta-Glo® Assay System, Promega Corp.), or adding(Z-DEVD)₂-rhodamine-110 (50 μM) to Beta-Glo® Buffer, or reconstitutingBeta-Glo® lyophilized substrate with Beta-Glo® Buffer and adding(Z-DEVD)₂-rhodamine-110 (50 μM). Caspase-3 (2 μl/ml, Pharmingen Corp),or β-galactosidase (0.1 μl/ml), or caspase-3 and β-galactosidase, werediluted in RPMI 1640 and 100 μl were added to wells of a 96-well whiteplate. 100 μl of the appropriate reagent were added to wells of a96-well plate and the plates were incubated at room temperature.Luminescence was measured using a DYNEX Laboratories MLX™ plateluminometer at 30 minutes. Fluorescence was measured 2 hours postincubation on a CytoFluor II Fluorescent plate reader with a filter setof 485_(EX)/530_(EM). All measurements were repeated at 18 hours withdifferent gain settings on the CytoFluor II fluorometer to compensatefor increased fluorescence.

Results

FIGS. 4A and C demonstrate the luminogenic assay for β-galactosidase isfunctional in the presence of the fluorogenic reagent to measurecaspase-3. FIGS. 4B and D demonstrate the fluorogenic assay to measurecaspase-3 is functional in the presence of the luminogenic reagent tomeasure β-galactosidase. As seen in FIGS. 4B and 4D, there was a minorcontribution of the luminogenic reagent components to backgroundfluorescence. However, there was almost no contribution of thefluorogenic reagent components to luminescence (FIGS. 4A and 4C).

E. Spectral Scans of Substrates for Luminogenic Assays

Luciferin, aminoluciferin, and Z-LETD-aminoluciferin, were diluted toapproximately 2 μM in a buffer containing 0.1M Tris pH 7.3, 2 mM EDTA,and 10 mM MgSO₄. Samples were scanned on a SPEX Fluorolog-2 spectrometerwith 1.25 mm excitation and emission slit filter present, at 1 nmwavelength interval and 0.2 second integration time. All scans wereperformed using a quartz cuvette.

Results

For luciferin and aminoluciferin, excitation was at 325 nm and emissionwas captured from 375 to 750 nm, and excitation was captured at 280-550nm with emission measured at 600 nm (FIGS. 5A and 5B). ForZ-LETD-aminoluciferin, excitation was at 325 nm and emission wascaptured between 375-750 nm, and excitation was captured at 280-500 nmwith emission measured at 525 nm (FIG. 5C). Interestingly, when apeptide was conjugated to aminoluciferin (FIG. 5C), the emission peak ofthe conjugate was blue shifted to shorter wavelengths. This wasunexpected and therefore allows for dual luminescence/fluorescentmeasurements, particularly when using a fluorophore that emits in thesame wavelength range as aminoluciferin emits.

EXAMPLE II Method to Detect False Results Methods

Caspase-Glo™ 3/7 Reagent (Caspase-Glo™ 3/7 Assay, Promega, Corp.) whichcontains Z-DEVD-aminoluciferin was combined with (Z-DEVD)₂-rhodamine-110or Ac-DEVD-AMC in the presence of caspase-3 with either a caspase-3inhibitor (Ac-DEVD-CHO, 10 μM) or with a luciferase inhibitor(Resveratol, 5 μM). The luminescent signal from caspase-3 cleavage ofZ-DEVD-aminoluciferin was read at 30 minutes, while the fluorescentsignals from caspase-3 cleavage activity were read at 2 hours using theappropriate AMC or rhodamine 110 filter sets.

Results

Luminescence gave the largest signal to background ratio, followed byrhodamine-110, then AMC (FIG. 6). All three substrates for detectingcaspase-3 were consistently and negatively impacted by the addition of aknown caspase-3 inhibitor. This suggests that luminogenic andfluorogenic reagents can be combined, e.g., to control for potentialfalse interferences when either assay is performed. Thus, multiplexedsignals can be used to determine if an agent is a true inhibitor of aparticular enzyme.

EXAMPLE III Additional Exemplary Multiplex Assays A. Multiplex Assay forLactate Dehydrogenase (LDH) and Adenosine Triphosphate (ATP) in a SingleWell Format

The following detection reagents were prepared: 1) LDH reagent (30 mMHEPES, pH 7.4, 10 mM NaCl, 20 mM MgSO₄, 250 μM resazurin (Aldrich)) wasused to reconstitute the lyophilized substrate component fromCytoTox-ONE™ Homogeneous Membrane Integrity Assay (Promega Corp,Technical Bulletin 306); 2) ATP reagent (30 mM HEPES pH 7.4, 10 mM NaCl,20 mM MgSO₄) was used to reconstitute the lyophilized substrate fromCellTiter-Glo™ Luminescent Cell Viability Assay (Promega Corp, TechnicalBulletin 288); 3) LDH/ATP combination reagent (30 mM HEPES pH 7.4, 10 mMNaCl, 20 mM MgSO₄, 250 μM resazurin (Aldrich)) was used to reconstitutethe lyophilized substrate component from CytoTox-ONE™, which in turn wasused to reconstitute the lyophilized substrate from CellTiter-Glo™.

Sample dilutions of LDH (0, 1:8000, 1:4000, 1:2000, diamonds), ATP (0,1.25, 2.5, and 5 μM, squares), and a combination of LDH/ATP (0/0 μM,1:8000/1.25 μM, 1:4000/2.5 μM, and 1:2000/5 μM, respectively, triangles)were made with a 10 mM HEPES pH 7.5, 0.1% Prionex (PentaPharma Corp)solution, and 100 μl of the dilutions (n=4) were added to wells of awhite, 96-well plate. The appropriate detection reagent (100 μl) wasadded to the samples, the plates were protected from light, mixed for 30seconds, and incubated at room temperature. Following an eight minuteincubation, fluorescence was measured on a Labsystems Fluoroskan Ascentplate reader with filter set 560_(EX)/590_(Em). At 30 minutespost-incubation luminescence was recorded using a Dynex MLX plateluminometer.

Results

There was a minor effect of LDH and its fluorogenic detection reagent onthe luminogenic assay for ATP (FIG. 7A) when compared to the controlreaction (FIG. 7C); however, detection of ATP was still possible. Theaddition of a luminogenic detection reagent to the fluorogenic assay forLDH did not affect background fluorescence (FIG. 7B), and althoughoverall fluorescence decreased when compared to the control reaction(FIG. 7D) LDH activity was still detectable.

B. Multiplex Assay for LDH and Caspase-3 in a Single Well Format

The following detection reagents were prepared: 1) LDHreagent—Caspase-Glo™ 3/7 Buffer supplemented with 238 μM resazurin wasused to reconstitute the CytoTox-ONE™ lyophilized substrate; 2)caspase-3 reagent—the Caspase-Glo™ 3/7 buffer was used to reconstitutethe Caspase-Glo™ 3/7 lyophilized substrate as per Promega TechnicalBulletin 323; 3) LDH/caspase-3 combined reagent—LDH reagent (prepared asabove) was used to reconstitute the lyophilized Caspase-Glo™ 3/7substrate. The LDH detection reagent compound of the LDH/caspase-3combined reagent is unstable due to the presence of DTT in theCaspase-Glo™ 3/7 lyophilized substrate, so this reagent was preparedimmediately prior to addition to samples.

Sample dilutions were prepared in 10 mM HEPES pH 7.5, 0.1% Prionex(PentaPharma Corp) solution: 0, 1:8000, 1:4000, 1:2000 dilutions of LDH(diamonds); 0, 5, 10, and 20 U/ml caspase-3 (BIOMOL Laboratories,squares), and a combination of LDH/caspase-3 (0/0 U/ml, 1:8000/5 U/ml,1:4000/10 U/ml, and 1:2000/20 U/ml, respectively, triangles). 100 μl ofthe dilutions (n=4) were added to white, 96-well plates. The appropriatedetection reagent (100 μl) was added to the samples, and the plates wereprotected from light, mixed for 30 seconds, and incubated at roomtemperature. Following a six minute incubation at room temperature,fluorescence was measured on a Labsystems Fluoroskan Ascent plate readerwith filter set 560_(EX)/590_(Em). At 45 minutes post-incubationluminescence was recorded using a Dynex MLX plate luminometer.

Results

There was a decrease in luminescence with the addition of a fluorogenicLDH detection reagent to a multiplex reaction when compared to a controlreaction (FIGS. 8A and 8C, respectively). Despite the decrease in totalluminescent signal in FIG. 8A, the luminescent caspase-3 assay wasfunctional in the presence of the fluorogenic LDH detection reagent.FIG. 8B shows there was an increase in fluorescence background when theluminogenic caspase-3 detection reagent was added to the multiplexreaction when compared to control (FIG. 8D); however, FIG. 8Ademonstrates the fluorogenic assay for LDH is functional in the presenceof the luminogenic detection reagent for caspase-3. There was nocontribution of LDH to background luminescence (FIG. 8C), and there wasno contribution of caspase-3 to background fluorescence (FIG. 8D).

C. Multiplex Assay for Caspase-3 and Protein Kinase A (PKA) in a SingleWell Format

The following detection reagents were prepared: 1) PKA reagent—a 1×reaction buffer was prepared which contains 100 mM Tris pH 7.3, 100 mMMgCl₂, 1:1000 dilution of a PKA rhodamine-110 substrate (ProFluor™ PKAAssay, Promega Corporation, Technical Bulletin 315), and 400 μM ATP; 2)caspase-3 reagent—a 1× reaction buffer was prepared containing 100 mMTris pH 7.3, 100 mM MgCl₂, 150 μg/ml recombinant thermostableluciferase, 80 μM Z-DEVD-aminoluciferin (Promega Corp), 400 μM ATP, 100μM DTT (Promega Corp), 2.5 mM CaCl₂ (Fisher), 40 mM MgSO₄ (Fisher), and0.2% Tergitol NP-9 (Sigma); 3) kinase/caspase-3 combined reagent—a 1×reaction buffer was prepared containing 100 mM Tris pH 7.3, 100 mMMgCl₂, 1:1000 dilution of a PKA rhodamine-110 substrate, 150 μg/mlrecombinant thermostable luciferase, 80 μM Z-DEVD-aminoluciferin, 400 μMATP, 100 μM DTT, 2.5 mM CaCl₂, 40 mM MgSO₄, and 0.2% Tergitol NP-9; 4)protein kinase stop reagent—a 1× stop reagent was prepared containing100 mM Tris pH 7.3, 100 mM MgCl₂, 1:50 dilution of protease reagent(ProFluor™ PKA Assay), 30 μM staurosporine (BIOMOL Laboratories).

Sample dilutions were prepared in 10 mM HEPES pH 7.5, 0.1% Prionex(PentaPharma Corp) solution; 0, 1, 2, and 4 U/ml PKA (diamonds), 0, 5,10, and 20 U/ml caspase-3 (squares), and a combination of PKA andcaspase-3 (0/0 U/ml, 1/5 U/ml, 2/10 U/ml, and 4/20 U/ml, respectively,triangles), and 40 μl of the dilutions (n=4) were added to white,96-well plates. The appropriate detection reagent (40 μl) was added tothe samples, the plates were protected from light, mixed for 30 seconds,and incubated at room temperature for 20 minutes. Following incubation,40 μl of a protein kinase stop reagent were added to the wells whichcontained either the kinase reagent alone or the combinationkinase/caspase-3 reagent. The plates were mixed an additional 30seconds, protected from light, and incubated for 30 minutes longer atroom temperature. Fluorescence was measured on a Labsystems FluoroskanAscent plate reader with filter set 485_(EX)/527_(Em). Luminescence wasrecorded using a Dynex MLX plate luminometer.

Results

The addition of a fluorogenic PKA assay detection reagent caused theluminescent background to increase (FIG. 9A) when compared to thecontrol reaction where PKA was present but the complete PKA detectionreagent was absent (FIG. 9C). However, reaction luminescence resultingfrom caspase-3 activity increased proportionately and the conditions didnot appear to affect the luminogenic caspase-3 reaction itself. Additionof the detection reagent for caspase-3 to the fluorogenic assay for PKA(FIG. 9B) decreased overall fluorescence by more than 50% when comparedto the fluorescent control reaction (FIG. 9D) where caspase-3 waspresent but the complete caspase-3 detection reagent was absent.Caspase-3 and PKA activities were measurable over background using thesemultiplex conditions.

D. Multiplex Assay for Renilla Luciferase and Caspase-3 in a Single WellFormat

The following detection reagents were prepared: 1) EnduRen™ (PromegaCorp.), a cell permeant modified coelenterazine substrate for Renillaluciferase, was diluted to 600 μM into F-12 tissue culture mediumsupplemented with 10% fetal bovine serum and 500 μg/ml G-418 sulfate; 2)caspase-3 substrate: (Z-DEVD)₂-rhodamine-110 (Promega Corp.) was dilutedto 250 μM into F-12 tissue culture medium supplemented with 10% fetalbovine serum and 500 μg/ml G-418 sulfate; 3) luciferase/caspase-3combined substrates: EnduRen™ (600 μM) and (Z-DEVD)₂-rhodamine-110 (250μM) were diluted in F-12 tissue culture medium supplemented with 10%fetal bovine serum and 500 μg/ml G-418 sulfate.

CHO—K1 cells (ATCC) which stably express Renilla luciferase (CHO—K1hRL25) were maintained in 10% fetal bovine serum and 500 μg/ml G-418sulfate and used for cell based experiments. Experimental conditionsutilizing these cells included: 1) varying levels of luciferase activitydue to addition of staurosporine, 2) varying levels of luciferaseactivity due to staurosporine addition with caspase-3 enzyme addition,and 3) luciferase activity with no staurosporine but with addition ofcaspase-3 enzyme.

CHO—K1 hRL25 cells were harvested and plated into a 96-well clearbottom, white walled tissue culture plate at a density of 20,000cells/well, and incubated overnight at 37° C. in 5% CO₂. Staurosporineat a final concentration of 0, 0.5, 1, 2 μM (10 μl/well) was added tothe appropriate wells to initiate cell death, thus altering luciferaseactivity. Cells were incubated for an additional 3.5 hours at 37° C. in5% CO₂. Various concentrations of caspase-3 (BIOMOL Laboratories) wereadded to the appropriate wells at 0, 5, 10, and 20 U/ml, in tissueculture medium (10 μl/well). Therefore, combined staurosporine/caspase-3concentrations for data points were 0 μM/0 U/ml, 0.5 μM/5 U/ml, 1 μM/10U/ml, and 2 μM/20 U/ml, respectively. Immediately after addition of thecaspase-3 enzyme, 10 μl/well of either luciferase substrate, caspase-3substrate, or luciferase/caspase-3 substrates were added to theappropriate wells. After addition of the detection reagents, the plateswere mixed briefly and incubated at 37° C. in 5% CO₂ for two hours.Fluorescence was measured on a Labsystems Fluoroskan Ascent plate readerwith filter set 485_(EX)/527_(Em). Luminescence was recorded using aDynex MLX plate luminometer.

Results

Activity of Renilla luciferase was used in these assays as an internalcontrol for cell death. Therefore, as staurosporine concentrationincreases, luciferase activity should decrease. FIG. 10A shows that theaddition of the caspase-3 substrate did not negatively affect theluciferase reaction when compared to the control reaction (FIG. 10C).FIG. 10B shows that the addition of the luciferase substrate had noeffect on background fluorescence, even though there was a slightincrease in total fluorescence when compared to FIG. 10D. Theluminogenic assay for Renilla luciferase was fully functional in thepresence of caspase-3 or the caspase-3 substrate, and the fluorogenicassay for caspase-3 was only slightly affected, but fully functional, inthe presence of Renilla luciferase or the Renilla luciferase substrate.

REFERENCES

-   Bronstein et al. (In: Bioluminescence and Chemiluminescence:    Molecular Reporting with Photons, pp. 451-457 (1996).-   DeJager et al., Clin. Diag. Lab. Immunolo., 10:133 (2003).-   Fernandes-Alnemri et al., PNAS USA, 93:7464 (1996).-   Gazi et al., Luminescent, 17:106 (2002).-   Liu et al., Luminescence, 15:45 (2000).-   Masuda-Nishimura et al., Lett. Appl. Microbio., 30:130 (2000).-   Miska and Geiger, J. Clin. Chem. Clin. Biochem., 25:23 (1989).-   Monsees et al., Anal. Biochem., 221:329 (1994).-   Monsees et al., J. Biolum. Chemilum., 10:213 (1995).-   Nicholson et al., Nature, 376:37 (1995).-   Nolkrantz et al., Anal. Chem., 7:4300 (2002).-   Qazi et al., Luminescence, 17:106 (2002).-   Tewari et al., Cell, 81:801 (1995).-   Thornberry et al., Nature, 356:768 (1992).

All publications, patents and patent applications are incorporatedherein by reference. While in the foregoing specification this inventionhas been described in relation to certain preferred embodiments thereof,and many details have been set forth for purposes of illustration, itwill be apparent to those skilled in the art that the invention issusceptible to additional embodiments and that certain of the detailsdescribed herein may be varied considerably without departing from thebasic principles of the invention.

1-48. (canceled)
 49. A method to detect the presence or amount of atleast two molecules in a sample, comprising: a) contacting the samplewith a first substrate for a first enzyme, a second substrate for asecond enzyme and optionally a third enzyme, wherein a reaction betweenthe first substrate and first enzyme or the third enzyme and a productof a reaction between the first enzyme and first substrate yields aluminogenic product, wherein the second substrate and/or a product of areaction between the second substrate and the second enzyme are notluminogenic, and wherein the first and second enzymes are not the same;and b) detecting the presence or amount of the first and second enzymesor a cofactor for a reaction mediated by the first or second enzyme. 50.The method of claim 49 wherein luminescence is detected.
 51. The methodof claim 49 wherein at least one enzyme is a protease.
 52. The method ofclaim 49 wherein the second enzyme does not react substantially with thefirst substrate.
 53. The method of claim 49 wherein the first enzymedoes not react substantially with the second substrate.
 54. The methodof claim 49 wherein the second substrate or the product of the reactionbetween second substrate and the second enzyme is fluorescent.
 55. Themethod of claim 49 wherein fluorescence is employed to detect thepresence or amount of the second enzyme or cofactor.
 56. The method ofclaim 49 wherein at least one enzyme is a caspase.
 57. The method ofclaim 49 wherein one of the substrates is a substrate for trypsin ortryptase.
 58. The method of claim 49 wherein the sample is a celllysate.
 59. The method of claim 58 wherein the sample is a cellularsample that is treated with a cell death inducing agent prior to lysis.60. The method of claim 49 wherein the sample comprises intact cells.61. The method of claim 49 wherein the third enzyme is a luciferase. 62.The method of claim 61 wherein the luciferase is a beetle luciferase.63. The method of claim 62 wherein the second substrate or the productof the reaction between second substrate and the second enzyme isfluorescent.
 64. The method of claim 49 wherein the sample is contactedwith the first substrate before the second substrate.
 65. The method ofclaim 49 wherein the sample is contacted with the second substratebefore the first substrate.
 66. The method of claim 49 wherein thesample is simultaneously contacted with the first and the secondsubstrates.
 67. The method of claim 49 wherein the presence or amount ofthe first enzyme or first cofactor is detected before the presence oramount of the second enzyme or second cofactor is detected.
 68. Themethod of claim 49 wherein the presence or amount of the second enzymeor second cofactor is detected before the presence or amount of thefirst enzyme or first cofactor is detected.
 69. The method of claim 68wherein the second substrate or the product of the reaction betweensecond substrate and the second enzyme is fluorescent.
 70. The method ofclaim 69 wherein the second substrate or the product of the reactionbetween the second substrate and the second enzyme comprises ethidiumbromide, fluorescein, Cy3, BODIPY, a rhodol, Rox, 5-carboxyfluorescein,6-carboxyfluorescein, an anthracene, 2-amino-4-methoxynapthalene, aphenalenone, an acridone, fluorinated xanthene derivatives, α-naphtol,β-napthol, 1-hydroxypyrene, coumarin, 7-amino-4-methylcoumarin (AMC),7-amino-4-trifluoromethylcoumarin (AFC), Texas Red,tetramethylrhodamine, carboxyrhodamine, or rhodamine, cresyl,rhodamine-110 or resorufin.
 71. The method of claim 49 wherein thecofactor is ATP.
 72. The method of claim 49 wherein the presence oramount of the second enzyme is detected by contacting the sample with afourth enzyme and third substrate for a reaction between the product ofthe reaction between the second substrate and second enzyme which yieldsa fluorogenic product. 73-76. (canceled)
 77. A kit comprising: anonluminogenic substrate; and a luminogenic substrate.
 78. The kit ofclaim 77 further comprising an enzyme capable of mediating aluminescence reaction with the luminogenic substrate.
 79. The kit ofclaim 77 further comprising instructions for conducting a luminogenicreaction and a nonluminogenic reaction in a single reaction vessel whichcomprises the nonluminogenic substrate and the luminogenic substrate.80. A kit comprising: a nonluminogenic substrate; and an enzyme capableof mediating a luminescence reaction.
 81. The kit of claim 80 furthercomprising a luminogenic substrate for the enzyme.
 82. The kit of claim77 or 80 wherein the nonluminogenic substrate is a fluorogenicsubstrate.
 83. The kit of claim 78 or 80 wherein the enzyme is aluciferase.
 84. The kit of claim 80 further comprising instructions forconducting a luminogenic reaction and a nonluminogenic reaction in asingle reaction vessel which comprises the nonluminogenic substrate andthe enzyme.